CN101407778A - Spherical bacillus and organic solvent-resistant stable proteinase produced thereby - Google Patents
Spherical bacillus and organic solvent-resistant stable proteinase produced thereby Download PDFInfo
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- CN101407778A CN101407778A CNA2008102344573A CN200810234457A CN101407778A CN 101407778 A CN101407778 A CN 101407778A CN A2008102344573 A CNA2008102344573 A CN A2008102344573A CN 200810234457 A CN200810234457 A CN 200810234457A CN 101407778 A CN101407778 A CN 101407778A
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Abstract
The invention discloses a bacillus sphaericus DS 11. The strain is a short rod shape Gram-positive bacteria, the growth temperature range of the strain is 4 to 35 DEG C and the optimal growth temperature is 28 DEG C; the growth pH range is between 4 and 11, and the optimal growth pH is 7; the growth NaCl concentration is between 0.3 and 10 percent, and the optimal NaCl concentration is 3 percent. The bacillus sphaericus DS 11 can endure an organic solvent with Log P of less than or equal to 2.0. The invention further discloses a method for producing an inactive protease of the organic solvent by utilizing the bacillus sphaericus DS 11 as well as the inactive protease product of the organic solvent which is obtained by the method, has stronger organic solvent tolerance and can be used for the synthesis of organic-phase high-performance catalytic polypeptide.
Description
Technical field
The present invention relates to obtain spherical bacillus DS11 (Bacillus sphaericus DS11) a kind of from the seawater in marine site, Yellow Sea of China Lianyun Harbour, the separation; The invention still further relates to the method and the product of this spherical bacillus product organic solvent stable protease.
Background technology
Proteolytic enzyme can be in the hydrolysis of water catalysis peptide bond, and the catalysis peptide bond is synthetic under the organic phase condition, is one of zymin the most widely at present, is widely used in that polypeptide is synthetic, fields such as protein processing, food, pharmacy, process hides and washing composition.Proteolytic enzyme requires proteolytic enzyme tolerance organic solvent when organic phase catalysis peptide is synthetic.But organic solvent has stronger toxic action to most of enzymes.Means such as the chemically modified by enzyme, embedding, fixing, protein engineering and orthogenesis can improve the tolerance of enzyme to organic solvent.But these method cost height, complicated operation if enzyme has high reactivity and stability under the situation that organic solvent exists, can be simplified working process greatly, reduce cost.
The organic solvent-resistant extreme microorganism be can be in the environment that the higher concentration organic solvent exists a quasi-microorganism of growth and breeding.The organic solvent-resistant extreme microorganism often produces organic solvent stability enzyme, as lipase, proteolytic enzyme and amylase.Therefore screen the organic solvent-resistant extreme microorganism and become the main means that obtain organic solvent stability enzyme.Only find that at present Pseudomonas aeruginosa and Bacillus sp. can produce organic solvent tolerant protease.And do not carry out suitability for industrialized production, be difficult to satisfy the needs in market.
Domestic less about organic solvent-resistant microorganism and organic solvent enzyme stability class research report, the at present domestic report that does not still have spherical bacillus to produce the organic solvent stable protease.Screen novel organic solvent stable protease,, satisfy the suitability for industrialized production needs and study proteolytic enzyme organic solvent stability mechanism laying the foundation for enriching organic solvent stable protease kind.
Summary of the invention
Technical problem to be solved by this invention is at the deficiencies in the prior art, provides a kind of and produces the method for organic solvent stable protease by spherical bacillus (Bacillus sphaericus DS11) DS11.
The invention also discloses the character that aforesaid method produces the organic solvent stable protease.
The invention also discloses the isolation cultivation method of spherical bacillus (Bacillus sphaericus DS11) DS11.
Biomaterial spherical bacillus DS11 (Bacillus sphaericusDS11) bacterial strain related among the present invention is a kind of disclosed bacterial strain, open on August 4th, 2008 at Genebank, the 16S rDNA sequence of this bacterial strain is also open at Genebank, its length is 1467bp, accession number is: EU835735, and network address is:
http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi？db＝nuccore&id＝194716764。
This bacterial strain is public material, rise in 20 years in this patent application day, the public if desired, Huaihai Institute of Technology oceanography institute laboratory can externally provide.
The present invention is its product machine solvent stability proteolytic enzyme and enzyme producing method of research on the basis of above-mentioned disclosed spherical bacillus DS11 (Bacillus sphaericus DS11) bacterial strain.
Technical problem to be solved by this invention is to realize by following technical scheme.The present invention is a kind of spherical bacillus DS11 (Bacillus sphaericus DS11), be characterized in, it adopts following method to carry out separation and Culture, adding the seawater sample 1ml that takes from marine site, Yellow Sea of China Lianyun Harbour to the 250ml triangular flask is equipped with in the MLB liquid nutrient medium that 50mL contains 10% benzene, clog bottleneck with rubber plug, 28 ℃, 160r/min is transferred in the new MLB liquid nutrient medium 3 times in this way after cultivating 4d; With gained nutrient solution dilution 10
5Doubly, draw 10 μ l coating MLB culture medium flat plate, in 28 ℃ of incubators, cultivate 2d, observe colonial morphology, line is purified to purebred back dibbling in the SMA culture medium flat plate, cultivate 2d in 28 ℃ of incubators, the ratio that preliminary screening goes out colony diameter and transparent circle diameter is greater than 5 bacterial strain, and the primary dcreening operation bacterial strain is inoculated into respectively produces the enzymic fermentation substratum, 28 ℃, after 180r/min cultivated 48h, 4 ℃ of centrifugal 10min of following 8000g measured the supernatant liquor protease activity; Get the 1mL supernatant liquor respectively and add 1mL benzene, at 30 ℃, detect proteinase activity behind the 180r/min oscillation treatment 1h, the minimum bacterial strain of protease activity reduction was spherical bacillus DS11 (Bacillussphaericus DS11) before and after benzene was handled.
The following evaluation that the contriver did about bacterial strain.
1.1 morphological specificity: Gram-positive, rod-short, size are 2.2-2.7 μ m * 0.7-1.3 μ m, do not have pod membrane, gemma are arranged.
1.2 the colony characteristics on skimming milk (SMA) solid medium: diameter 2mm-4mm is white, moistening, the edge is smooth, center protrusion, easy picking, produces the obvious transparent circle.
1.3 physiological and biochemical property:
Physiological and biochemical property: this bacterium catalase, urase, MR test all positive, and oxydase, VP test negative, can not produce indoles, do not produce H2S; Can the hydrolysis gel, tween-80, triolein, casein, can not hydrolyzed starch.Bacterial strain DS11 can utilize glucose, sucrose, wood sugar, N.F,USP MANNITOL, can not utilize maltose, lactose, raffinose, rhamnosyl, propanedioic acid to be carbon source.Passing through Bergey ' s Manual of Systematic Bacteriology Analysis and Identification spherical bacillus DS11 (Bacillus sphaericus DS11), to belong to this bacterium of Bacillus Sp. preliminary evaluation be bacterium spherical bacillus (Bacillus sphaericus).
1.4 growth characteristics
The growth temperature range of bacterial strain is 4-36 ℃, and optimum growth temperature is 28 ℃ (Fig. 1); Growth pH scope is 4-11, and the suitableeest growth pH is 7 (Fig. 2); The NaCl concentration of growth is 0.3%-10%, and the suitableeest NaCl concentration is 3% (Fig. 3).Can tolerate polarity constant (Log P) is lower than and equals 2.0 organic solvent.
The physiological and biochemical property of table 1 bacterial strain DS11
"+" is positive, and "-" is negative,
1.4 the molecular biology identification of bacterial strain DS11
16S rDNA pcr amplification and sequential analysis: extract dna profiling with the Takara test kit, and in-40 ℃ of preservations.Forward primer P1:5 '-GAGAGTTTGATCCTGGCTCAG-3 ', reverse primer P2:5 '-CGGCTACCTTGTTACGAC-3 '.Reaction system 50 μ l, Taq enzyme 2U, reaction conditions are 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, and 54 ℃ of annealing 40s, 72 ℃ are extended 1min, 35 circulations, 72 ℃ are extended 7min.The purifying of PCR product, clone, order-checking are finished by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.The 16S rDNA of amplification bacterial strain GS230, its length is 1467bp.This sequence is submitted GenBank (accession number: EU835735) and with the sequence in the GenBank database carry out homology relatively, find that the 16S rDNA of bacterial strain DS11 and Bacillus birdss of the same feather flock together naturally.Bacterial strain and DS11 that selection and its homology are high carry out phylogenetic structure, (16S rDNA GenBank accession number: AB116123) sibship is nearest, identifies that further this bacterial strain is spherical bacillus Bacillus sphaericus to draw bacterial strain DS11 and Bacillussphaericus.
Produce the method for organic solvent stable protease about bacterial strain of the present invention.
1.1 relevant substratum among the present invention
Skimming milk (SMA) solid medium (g/L): peptone 5.0, yeast powder 3.0, skim-milk 25.0, agar 40, Chen Haishui 1000.0ml, pH 7.0.
MLB liquid nutrient medium (g/L): peptone 10.0, yeast powder 5.0, NaCl 10.0, MgSO
47H
2O 0.5, and pH 7.0.
MLB solid medium (flat board) is (g/L): peptone 10.0, and yeast powder 5.0, NaCl 10.0, MgSO
47H
2O 0.5, agar 40, and pH 7.0.
Produce enzymic fermentation substratum (g/L): peptone 10.0, (NH
4)
2SO
41.0, KH
2PO
40.5; MgSO
47H
2O 0.3, CaCl
22H
2O 1.0, and NaCl 1.0, glycerine 10.0ml, and pH 7.0.
1.2 enzymatic production
Spherical bacillus DS 11 (Bacillus sphaericus DS11) inoculation is arrived the MLB liquid nutrient medium, 180r/min, cultivate 24h as seed liquor for 28 ℃, seed liquor is inoculated in 1% inoculum size produces the enzymic fermentation substratum, 180r/min, cultivate 32h for 28 ℃, 10, the gained supernatant liquor is crossed 0.22 μ m filter membrane and is organic solvent stable protease crude enzyme liquid behind the centrifugal 20min of 000g.
Organic solvent tolerance about spherical bacillus DS11 (Bacillus sphaericus DS11) bacterial strain.
Spherical bacillus DS11 (Bacillus sphaericus DS11) inoculation is arrived MLB solid medium flat board, cover the organic solvent of 7.0ml opposed polarity constant on the flat board, seal with preservative film and to prevent organic solvent volatilization, cultivate 48h for 28 ℃, observe the strain growth situation.The result shows, spherical bacillus DS11 (Bacillus sphaericus DS11) can be not more than on the flat board that 2 organic solvent covers at the polarity constant and grow, and forms bacterium colony.See Table 2.
Table 2 organic solvent is to the influence of strain growth
"+" is growth, and "-" be not for growing
About the influence of organic solvent to organic solvent stable protease stability.
1, organic solvent is to the influence of organic solvent stable protease stability.
The organic solvent that adds the opposed polarity constant in the 3.0ml crude enzyme liquid is added 1.0ml, 30 ℃, 140r/min, behind the 14d, the result shows, the residual enzyme after methyl alcohol, ethanol, 1-butanols are handled live respectively 1 for protoenzyme alive 35%, 36% and 42%; In benzene, dimethylbenzene, hexane, normal hexane, keep the enzymic activity more than 82%; N-decane, octane, octane-iso, heptane etc. can improve the activity of enzyme.See Table 3.
Table 3 organic solvent is to the influence of proteolytic enzyme stability
2, organic solvent stable protease determination of activity.
2.0ml is contained the caseic Tris-HCl damping fluid of 2% (w/w) (50mM, pH 8.0) and 37 ℃ of preheating 10min of crude enzyme liquid of 2.0ml dilution after mix, 37 ℃ of waters bath with thermostatic control reaction 10min add 4.0ml TCA solution (0.11M trichoroacetic acid(TCA), 0.22M acetate is received, 0.33M acetate), behind 4 ℃ of placement 20min, 15, the centrifugal 15min of 000g, supernatant liquor is measured the 280nm absorbance value, and under condition determination, the enzyme amount that per minute catalysis produces 1 μ g tyrosine is defined as 1 enzyme unit (U) alive.
3, the application of organic solvent stable protease.The ability of tolerance organic solvent that spherical bacillus DS11 of the present invention (Bacillussphaericus DS11) bacterial strain produces the organic solvent stable protease is stronger, it is the same with other known organic solvent stable protease, can be used for the synthetic of organic phase efficient catalytic polypeptide.
Description of drawings
Fig. 1 is the influence figure of temperature and time to strain growth.
Fig. 2 is the influence figure of initial pH to strain growth.
Fig. 3 is the influence figure of NaCl to strain growth.
Embodiment
Embodiment 1.The separation and Culture of spherical bacillus DS11 (Bacillus sphaericus DS11) bacterial strain.
Adding the seawater sample 1ml that takes from marine site, Yellow Sea of China Lianyun Harbour to the 250ml triangular flask is equipped with in the MLB liquid nutrient medium that 50mL contains 10% benzene, clog bottleneck with rubber plug, 28 ℃, 160r/min is transferred in the new MLB liquid nutrient medium 3 times in this way after cultivating 4d; With gained nutrient solution dilution 10
5Doubly, draw 10 μ l coating MLB culture medium flat plate, in 28 ℃ of incubators, cultivate 2d, observe colonial morphology, line is purified to purebred back dibbling in the SMA culture medium flat plate, cultivate 2d in 28 ℃ of incubators, the ratio that preliminary screening goes out colony diameter and transparent circle diameter is greater than 5 bacterial strain, and the primary dcreening operation bacterial strain is inoculated into respectively produces the enzymic fermentation substratum, 28 ℃, after 180r/min cultivated 48h, 4 ℃ of centrifugal 10min of following 8000g measured the supernatant liquor protease activity; Get the 1mL supernatant liquor respectively and add 1mL benzene, at 30 ℃, detect proteinase activity behind the 180r/min oscillation treatment 1h, the minimum bacterial strain of protease activity reduction was spherical bacillus DS11 (Bacillus sphaericus DS11) before and after benzene was handled.
Embodiment 2.Spherical bacillus DS11 (Bacillus sphaericus DS11) strain characteristics.
This bacterial strain is Gram-positive, rod-short, the big or small 2.2-2.7 μ m * 0.7-1.3 μ m of being, does not have pod membrane, gemma is arranged.Colony diameter 2mm-4mm on skimming milk (SMA) solid medium is white, moistening, the edge is smooth, center protrusion, easy picking, produces the obvious transparent circle.This bacterium catalase, urase, MR test all positive, and oxydase, VP test negative, can not produce indoles, do not produce H2S; Can the hydrolysis gel, tween-80, triolein, casein, can not hydrolyzed starch.Bacterial strain DS11 can utilize glucose, sucrose, wood sugar, N.F,USP MANNITOL, can not utilize maltose, lactose, raffinose, rhamnosyl, propanedioic acid to be carbon source.
Embodiment 3.Spherical bacillus DS11 (Bacillus sphaericus DS11) bacterial strain organic solvent-resistant characteristic research.
Embodiment 4.Spherical bacillus DS11 (Bacillus sphaericus DS11) produces the method for organic solvent stable protease.
With embodiment 1 described spherical bacillus DS11 (Bacillus sphaericus DS11) bacterial classification inoculation to the MLB liquid nutrient medium, 180r/min, cultivate 24h as seed liquor for 28 ℃, seed liquor is inoculated in 1% inoculum size produces the enzymic fermentation substratum, 180r/min, cultivate 32h for 28 ℃, 10, the gained supernatant liquor is crossed 0.22 μ m filter membrane and is organic solvent stable protease crude enzyme liquid behind the centrifugal 20min of 000g.
Embodiment 5.Organic solvent is to the influence of embodiment 4 described organic solvent stable protease stability.
Get organic solvent stable protease crude enzyme liquid with 30 ℃ of the organic solvents of 25% (V/V) opposed polarity constant, 140r/min, handle 14d after, the residual enzyme work after methyl alcohol, ethanol, 1-butanols are handled be respectively that protoenzyme lives 35%, 36% and 42%; In benzene, dimethylbenzene, hexane, normal hexane, keep the enzymic activity more than 82%; N-decane, octane, octane-iso, heptane etc. can improve the activity of enzyme.
Claims (5)
1. a spherical bacillus DS11 (Bacillus sphaericus DS11), it is characterized in that, it adopts following method to carry out separation and Culture, adding the seawater sample 1ml that takes from marine site, Yellow Sea of China Lianyun Harbour to the 250ml triangular flask is equipped with in the MLB liquid nutrient medium that 50mL contains 10% benzene, clog bottleneck with rubber plug, 28 ℃, 160r/min is transferred in the new MLB liquid nutrient medium 3 times in this way after cultivating 4d; With gained nutrient solution dilution 10
5Doubly, draw 10 μ l coating MLB culture medium flat plate, in 28 ℃ of incubators, cultivate 2d, observe colonial morphology, line is purified to purebred back dibbling in the SMA culture medium flat plate, cultivate 2d in 28 ℃ of incubators, the ratio that preliminary screening goes out colony diameter and transparent circle diameter is greater than 5 bacterial strain, and the primary dcreening operation bacterial strain is inoculated into respectively produces the enzymic fermentation substratum, 28 ℃, after 180r/min cultivated 48h, 4 ℃ of centrifugal 10min of following 8000g measured the supernatant liquor protease activity; Get the 1mL supernatant liquor respectively and add 1mL benzene, at 30 ℃, detect proteinase activity behind the 180r/min oscillation treatment 1h, the minimum bacterial strain of protease activity reduction was spherical bacillus DS11 (Bacillus sphaericus DS11) before and after benzene was handled.
2. a kind of spherical bacillus DS11 according to claim 1 (Bacillus sphaericusDS11) is characterized in that, gained spherical bacillus DS11 (Bacillus sphaericusDS11) bacterial strain has following feature:
1) growth characteristics: the growth temperature range of bacterial strain is 5-45 ℃, and optimum growth temperature is 28 ℃; Growth pH scope is 4-11, and the suitableeest growth pH is 7; The NaCl concentration of growth is 0.3%-10%, and the NaCl concentration of suitable growth is 3%;
2) thalline feature: Gram-positive, rod-short, size are 2.2-2.7 μ m * 0.7-1.3 μ m, do not have pod membrane, gemma are arranged;
3) colony characteristics on the defatted milk solid substratum: diameter 2mm-4mm is white, moistening, the edge is smooth, center protrusion, easy picking, produces the obvious transparent circle;
4) physiological and biochemical property: this bacterial strain catalase, urase, MR test all positive, and oxydase, VP test negative, can not produce indoles, do not produce H
2S; Can the hydrolysis gel, tween-80, triolein, casein, can not hydrolyzed starch; Can utilize glucose, sucrose, wood sugar, N.F,USP MANNITOL, can not utilize maltose, lactose, raffinose, rhamnosyl, propanedioic acid to be carbon source.
3. a kind of spherical bacillus DS11 according to claim 1 (Bacillus sphaericusDS11), gained spherical bacillus DS11 (Bacillus sphaericus DS11) bacterial strain can tolerate the polarity constant and be not more than 2.0 organic solvent on the MLB solid plate.
4. method of producing the organic solvent stable protease as any one described spherical bacillus DS11 (Bacillussphaericus DS11) among the claim 1-3, it is characterized in that, its step is as follows, spherical bacillus DS11 (Bacillus sphaericus DS11) is inoculated in the MLB liquid nutrient medium, 180r/min, cultivate 24h for 28 ℃, as seed liquor; Seed liquor is inoculated in 1% inoculum size produces the enzymic fermentation substratum, 180r/min cultivates 32h for 28 ℃, and 10, the gained supernatant liquor is crossed 0.22 μ m filter membrane and is promptly got organic solvent stable protease crude enzyme liquid behind the centrifugal 20min of 000g.
5. a method as claimed in claim 4 is produced the organic solvent stable protease, it is characterized in that, described organic solvent stable protease has following character, in crude enzyme liquid, add 25% different organic solvent, 30 ℃, 140r/min, behind the 14d, the residual enzyme work after methyl alcohol, ethanol, 1-butanols are handled is respectively 35%, 36% and 42% of protoenzyme work; In benzene, dimethylbenzene, hexane, normal hexane, keep the enzymic activity more than 82%; N-decane, octane, octane-iso, heptane can improve the activity of enzyme.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643745A (en) * | 2012-04-27 | 2012-08-22 | 淮海工学院 | Method for rapidly screening secreted microorganism of organic solvent resistant protease |
| CN102864106A (en) * | 2012-09-24 | 2013-01-09 | 国家海洋局第二海洋研究所 | Ocean catalase production bacteria and directional screening method |
| CN103740605A (en) * | 2013-10-31 | 2014-04-23 | 华南理工大学 | Bacillus aquimaris and application thereof |
-
2008
- 2008-11-19 CN CNA2008102344573A patent/CN101407778A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643745A (en) * | 2012-04-27 | 2012-08-22 | 淮海工学院 | Method for rapidly screening secreted microorganism of organic solvent resistant protease |
| CN102643745B (en) * | 2012-04-27 | 2013-07-31 | 淮海工学院 | Method for rapidly screening secreted microorganism of organic solvent resistant protease |
| CN102864106A (en) * | 2012-09-24 | 2013-01-09 | 国家海洋局第二海洋研究所 | Ocean catalase production bacteria and directional screening method |
| CN102864106B (en) * | 2012-09-24 | 2014-04-23 | 国家海洋局第二海洋研究所 | Ocean catalase production bacteria and directional screening method |
| CN103740605A (en) * | 2013-10-31 | 2014-04-23 | 华南理工大学 | Bacillus aquimaris and application thereof |
| CN103740605B (en) * | 2013-10-31 | 2017-01-18 | 华南理工大学 | Bacillus aquimaris and application thereof |
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Open date: 20090415 |